RU2104039C1 - Live skin substituent, method of its preparing and method of live skin lesion treatment - Google Patents

Abstract

FIELD: medicine, implantology. SUBSTANCE: invention proposes live skin substituent for treatment of part or all layer of damaged skin, for example, at burns and wounds. Substituent consists of suspension covered with microsphere cells that can be applied on damaged skin similarly to paste or ointment. Skin implant can be adopted to counter alternations of skin lesions and does not require any fixation. Microspheres can be made of different biocompatible and in vivo resorbing materials. EFFECT: enhanced effectiveness of substituent. 26 cl, 12 dwg

Description

 The invention relates to tissue implants, in particular to the application of skin implants to treat all or part of the affected skin layer, for example, burns or other wounds.

 Damage to all or part of the skin layer, for example, for burns and other wounds, is a significant cost to the healthcare system. For example, about two million people in North America suffer burns every year. Of these, about 200,000 people are hospitalized, 15,000 of whom die due to burns. The total cost of treating such patients in the hospital is about $ 1,000 per 1% of the burn area (SU. S. 1992), so the average patient with burns, ranging from 20 to 30% of the total body skin area, accounts for about $ 25,000 ., not including the cost of further treatment and potential loss of productivity and income. For example, McMillan et. al (J. Burn Care Rehab, 6: 444-446; 1985) shows that surgery costs increase in a logarithmic relationship to the percentage of skin burn area. Clearly, there is a need for the development of technology that would reduce these costs and alleviate the suffering of patients.

 The skin consists of a dermal layer, which is located under the epidermal layer. The dermal layer consists mainly of fibroblasts and is approximately five times thicker than the epidermal layer. The epidermal layer of healthy skin, consisting mainly of keratinocytes and immune cells, such as dendritic langerans cells, prevents the loss of water and the penetration of microbes. Therefore, damage to part or all of the skin layer can threaten a person’s life. Thus, the speed of wound healing, preventing the loss of vital organization fluids and the penetration of bacteria, is a vital factor in the patient's recovery. Accordingly, a wide range of coatings for wounds has been developed in order to accelerate their healing.

 An existing method for healing burns and wounds involves using a patient’s own skin or tissue obtained from a deceased or a pig to transplant onto a patient’s wound. The traditional technology for skin grafts received from a patient (auto transplant) is usually very painful for a patient already suffering from a burn or other wound. An auto transplant consists of a significant thickness of both the epidermal and dermal layers of the skin taken from another part of the body. In order to reduce the amount of skin taken from another part of the body and, consequently, the size of a new wound, the autotransplanted tissue is treated in such a way as to form a lattice pattern along the skin lesion. However, the lattice sample in the autologous dermis layer is then filled with permanent scar tissue in vivo. These scars are often very large and can be very shapeless or, depending on their location, can cause dysfunction. In addition, the patient may not have enough unburnt surface for transplanting a sufficiently large amount of tissue transplanted to another place.

 A skin transplant obtained from a deceased (allotransplantation) or from a pig (xenotransplantation) is used to reduce the patient’s suffering and to promote wound healing. The main disadvantage of allotransplantation is the possibility of transmission of the disease (for example, HIV, hepatitis B). Moreover, the epidermal layer exhibits noticeable antagonism, as a result of which transplanted tissue, including allotransplantation, which is not obtained directly from the patient, is usually rejected within two weeks from the moment of transplantation. Although xenotransplantation is a type of transplant that saves human donor tissue, the tissue is rejected even faster than with allotransplantation, and it must be removed on the third day after application before drying and scab formation and before strong adhesion to the wound, causing the need for surgery.

 All these types of skin grafts, namely auto transplant, allotransplant and xenotransplant, are usually very thin and fragile, which makes it very difficult to transport and handle them. In addition, the transplanted tissue is fixed on the wound very often through extensive surgical sutures and / or staples, which significantly increases the patient's discomfort. In addition, patients with severe burns or wounds are already at risk, making surgery under anesthesia even more difficult and life-threatening.

 In later developments, for example, in US patent N 4996154 (publ. February 26, 1991, addressed to Millipore Corp., USA) and Beumar G.J. et. al. ("Biocompatibility and Characterization of a Polymeric Cell-Seeded Skin Substitute", 17th Ann Meeting Soc for Biomaterials; May 1-5, 1991, Scottsdale, Arizona, USA, p.263) it is proposed to grow skin cells on sheets with cells for protein implant. Sheets are sewn or brackets on the wound area, and the material of the temporary carrier substrate is usually resorbed by the body. Preliminary studies show that scar tissue growth can be reduced. In addition, the area of the cellular matrix is more stable than transplanted skin.

 However, there are many problems with planar cellular films or sheets of regenerated skin. Patches of cellular matrix skin typically deliver about 10 cm wide by 10 cm long and only a few cells thick. The specialist understands that burns often have a large and rarely uniform depth or planar structure. Patches inadequately correspond to contour changes in the skin, due to which, nevertheless, to a greater or lesser extent there is a problem of disfigurement. Being more stable than transplants, thin areas are still very fragile and in many cases thin sheets are strengthened for surgical procedures with a metal cloth impregnated with oil jelly. In addition, small pieces still have to be sewn and / or stapled to each other and to the body, which leads to an extension of the surgical procedure by a week and poses a risk to the patient.

 Moreover, the applied areas of the skin have very poor characteristics of gas and mass transfer, which leads to necrosis of the tissue as a result of insufficient nutrition of cells. This, in turn, can cause tissues to form blisters. The processes of gas and mass transfer can also be deteriorated due to the residual layer of oil jelly after removal of metal tissue with a subsequent surgical procedure. A residual layer of petroleum jelly can even lead to the release of various factors, such as growth factors, into this layer, where they will then not be able to act on the cells.

 Another drawback is the actual development of skin-derived cells in vitro. Typically, these fixed cells multiply under static conditions in a tissue culture vessel containing a cellular matrix in a relatively small volume of the culture medium and, thus, conditions are created for passive gas transmission from the surface of the medium to cells located on the bottom of the vessel. Much attention is paid to the consumption of nutrients from the environment, which may be accompanied by the formation of a microenvironment in the immediate vicinity of the cells. Such a microenvironment contains even less nutrients and tends to increase the concentration of metabolic intermediates, which negatively affects the development of cells. To overcome these problems, many laborious operations are required to change the environment, and each operation increases the possibility of microbial infection.

 In order to increase gas and mass transfer, a culture system was proposed that was enclosed in a gas-permeable bag with recirculating sediment (Marrow-Tech lnc., USA) to circulate the medium over the surface of the developing culture on the mesh tissue contained inside the bag. Cells are usually seeded randomly on a cell, resulting in many cell development centers. However, any cell that is not firmly attached to the surface can be circulated through the pump and experience shear and other destructive effects. The system is still laborious and cumbersome, which leads to an increase in the likelihood of infection due to excessive requirements for handling it. If the system is not static, then the medium is not completely homogeneous and the cells fixed on the cell are not in the same development phase due to the semi-static conditions of cultivation and random seeding of cells on the cell. In addition to the problem of homogeneity within one area, there is also the problem of homogeneity from site to site over the usually wide area of skin damage. Moreover, the effective area of viable cells can be significantly reduced by stapling and / or stapling along the edges of the plot, which can destroy or disrupt cell growth.

 None of the previously known technologies provides compliance with changes in the contour of damaged skin. Rarely, when damaged skin has a shape in perfect accordance with known planar transplants and areas, which leads to the possible formation of non-contact areas in which new skin does not quite correspond to them.

Moreover, many of these developments do not meet the extreme need for hardening of the epidermis layer. Water vapor passes through intact skin at a speed of about 8.5 g / m ² • h, and in an area without an epidermal layer, at a speed of about 150 g / m ² • h. Ideally, the permeability of the skin implant should be close to that of healthy skin to prevent tissue drying and thrombosis when the permeability is too high, and to prevent fluid accumulation and low adhesion of the transplanted tissue in the case of low permeability.

 Demetriou A. A. ("Replacement of liver function in rats by transplantation of microcarrier-attached hepatocytes" Science 233: 1190-1192; September 12, 1986) describes the attachment of hepatocytes to microcarriers from cross-linked dextran with a collagen coating for subsequent implantation in the region of rat peritoneum. Microcarriers are used as a surface for fixing, so that hepatocytes survive and act in vivo. Implanted microcarriers are not resorbed and not destroyed.

 International application PCT / US90 / 02257 (Vacanti et. Al., Published November 1, 1990, WO 90/12604) relates to an implant of a large volume of cells on a polymer matrix. The matrix is a fibrous biocompatible collapsible or non-degradable web-like material with gaps of 100-200 microns. Vacanti et. al. describes the fixation and development of hepatocytes on the matrix for subsequent implantation into the mesentery of the small intestine.

 The objective of the invention is the creation of a substitute for healthy skin for the treatment of damage to part or all of the skin layer, such as burns and injuries, which can adapt to changes in the contour, as well as the creation of an implant having a dermal layer and functioning epidermis and not requiring stitching, bracing or other methods .

 According to one aspect of the invention, there is provided a substitute for healthy skin for a fully or partially damaged skin layer, characterized in that it comprises a plurality of microspheres of biocompatible and resorbable in vivo material and a skin cell culture covering the microspheres, wherein the microspheres coated with skin cells are applied to the damaged skin.

 According to another aspect of the invention, a method for producing a substitute for healthy skin is provided, which consists in cultivating skin cells, producing a plurality of biocompatible resorbable microspheres and multiplying the attached skin cells in a medium until they merge or approximate merge, followed by concentration of the microspheres coated with cells to suspension by removing part or almost all medium and applying a suspension of cell-coated microspheres to the area of skin damage.

 Figure 1 shows photomicrography of attached dermal fibroblasts on microspheres 1 h after cell seeding; figure 2 - photomicrography of dermal fibroblasts attached to the same microspheres as in figure 1, 22 hours after plating of cells; figure 3 - schematically damage the entire layer of skin; in FIG. 4 is a schematic illustration of the damage to the entire skin layer according to FIG. 3 with the implantation of a healthy skin substitute according to the invention; figure 5 - photomicrography of keratinocytes attached to the microspheres, 24 hours after plating of cells; 6 is a photomicrograph of keratinocytes attached to microspheres, 24 hours after plating of cells, microspheres are incubated in culture medium for 24 hours before plating of cells; Fig.7 is a histogram of data obtained on a cytofluorometer; on Fig - photomicrography of dermal fibroblasts attached to processed by a fixing factor microspheres, 1 hour after plating of cells; Fig.9 is a photomicrograph of dermal fibroblasts attached to microspheres treated with a fixing factor, according to Fig.8 after 22 hours after plating of cells; figure 10 - photomicrography of dermal fibroblasts attached to the surface of the glass (control experiment); 11 is a photomicrograph of dermal fibroblasts attached to the planar surface of PMB-PHV (copolymer of polyhydroxybutyrate with polyhydroxyvalerate); on Fig - photomicrography of dermal fibroblasts attached to the plastical surface of PMB-PHV treated with a fusing factor.

 According to the invention, the cells are fixed on microspheres for subsequent implantation in vivo. In particular, a suspension of microspheres coated with skin cells is used as a substitute for healthy skin in case of damage to part or all of the skin layer. A suspension of microspheres coated with skin cells is applied directly to damaged skin in the same way as a solution or ointment.

 Skin cells can be obtained from a patient from a relatively small tissue explant. Alternatively, dermal fibroblasts exhibiting low allergenicity in transplants can be obtained from a donor, including a deceased. The epidermal layer shows significant antigenicity, therefore, implants from this layer usually need to be obtained directly from the patient. Studies show, however, that langerans cells are largely responsible for the antigenicity of the epidermal layer (Bagot M. et. Al., Clin Exp. Jmmunol. 71: 138; 1988). Therefore, it is possible to obtain a pure keratinocyte culture from a donor, as a result of which rejection will be either minimal or non-existent.

According to the invention, a small tissue explant, usually 4 cm ² in size, is obtained from a patient or donor using a dermatome or other suitable surgical instrument. The explant may include only the epidermal layer or a combination of the epidermal and dermal layers. The tissue is then destroyed by the action of a suitable enzyme by known technology and the cells are seeded in a tissue culture flask for subsequent cell growth of fibroblasts and / or keratinocytes. Cells of the dermal and epidermal layers can be isolated by enzymes such as dispase, either by impregnation with culture medium or phosphate buffered saline, or other well-established methods. For specialists, it is clear that a “bank” of cells obtained from a donor, including a deceased, can be created for timely treatment of skin lesions.

 The microspheres have a diameter in the range of about 50 to 500 microns, and preferably in the range of about 80 to 250 microns. Microspheres can be made of various materials, as will be discussed in more detail below. An important role in the choice of material is played by biocompatibility and the ability to resorb in the body without the formation of toxic by-products. One such suitable material is polyhydroxybutyrate (PHB), which is commonly used to make resorbable surgical staples or suture materials. PHB is resorbed in vivo to form carbon dioxide and water as end products. Other suitable materials are polylactide glycolides, which are commercially available for drug delivery systems. The polymers are absorbed by irregular hydrolysis of ester bonds and decomposed into lactonic and glycolic acids, which are common metabolic by-products.

According to one embodiment of the invention, dermal fibroblasts isolated from a tissue explant obtained from a patient or donor are cultured in a tissue culture flask until a sufficient number of cells is obtained to achieve a viable density in the bioreactor of about or at least 10 ⁴ up to 10 ⁵ cells / ml,
With a typical loading of microspheres from 3 to 5 mg / l, which corresponds to about 5 • 10 ⁶ microspheres / g dry weight, the cell density corresponds to about 5 cells / microsphere.

 After obtaining a sufficient number of cells, they are separated from the bottom of the tissue culture flask using, for example, trypsin. It will be apparent to one skilled in the art that, to obtain the required number of cells, a certain number of passages are required in tissue flasks of various sizes. After this, the cells are seeded in a bioreactor containing a suitable culture medium and microspheres at a density of 1 to 25 g / l, optimally 2-5 g / l. The microspheres can be added to the culture medium either immediately before seeding, or the microspheres are preliminarily impregnated in the medium before seeding for some time. Then the cells are fixed on the microspheres for some time under static or semi-static conditions, for example, for 3-6 hours

 The fixing step can be carried out in a reduced volume of medium with intermittent stirring for several minutes every half hour for 3-6 hours.

 In FIG. Figure 1 shows photomicrography showing dermal fibroblasts obtained from newborn placenta (neo-natal foreskin) attached to microspheres from PHB-PHV (polyhydroxybutyrate-polyhydroxyvaleriate copolymer), 1 h after cell plating. Dermal fibroblasts begin to attach to the microspheres.

 After some time, sufficient to further fix the cells on the microspheres, add the remaining amount of medium to create the desired working volume with subsequent continuous stirring. Figure 2 presents photomicrography, which shows dermal fibroblasts attached to microspheres from PHB-PHV, according to figure 1 after 22 hours after seeding. Most dermal fibroblasts attached to microspheres have turned from spherical to flat.

Alternatively, cells isolated from a tissue explant can be seeded directly in the bioreactor. For example, from a tissue explant with a size of 4 cm ² receive (2-5) • 10 ⁷ cells, which is enough to achieve a seeding density of 3 to 5 cells / microsphere in 1 l of medium.

Dermal fibroblast cells attached to the microspheres are then grown in culture suspension in a bioreactor, which usually has a working volume of 250 ml to several liters. It is possible to obtain a real skin implant using cells cultured in a 1 L bioreactor. With an average surface area of 5000 cm ² / g dry weight of the microspheres and a density of microspheres of 3 to 5 mg / ml, an effective surface area of 2 to 2.5 m ² per 1 liter of working volume is obtained. Cells eventually cover this entire surface, creating a comparable layer of skin for further migration and reproduction in vivo.

Smaller burns can be treated with culture from a smaller vessel, including a 250 mm spinning flask. A sophisticated bioreactor with close monitoring and control of parameters can be used. For example, such as described in US Pat. No. 4,906,577 (Armstrong, Fleming & Grenzowski), published March 6, 1990. Alternatively, a simple container with agitation in a CO ₂ incubator can be used. From the beginning of cell seeding to the final stage of unloading the cell-coated microspheres from the bioreactor inside the vessel, sterility is maintained in a known manner.

Like the static or semi-static cell culture technology practiced for the cell matrix regions described above, growing cells in suspension in a bioreactor gives higher cell productivity. This has been proven by the development of a suspension of cell culture, mounted on microspheres, representing small balls with a diameter of 100 to 200 μm with a surface area of about 5000 cm ² / g, usually from crosslinked dextran. Certain rows of cells were grown on microcarriers to prove the productivity of the element upon cell growth and / or product formation exceeding that achieved by static cultivation in a tissue culture flask (van Wezel, AL "Growth of Cell Strains and Primary Cells in Microcarriers in Homogeneous Culture" Nature 216: 64-65; October 7, 1967).

Static cultivators for producing skin grafts on cellular sheet substrates in tissue culture containers have an effective surface / volume (S / V) ratio of about 2-5 cm ^-1 in comparison with an S / V ratio of about 150 cm ^-1 for microspheres suspended at a concentration of 25 g / l. Moreover, suspension cultivation is less time consuming and the culture is more homogeneous, due to which the problems of gas transmission, depletion of the nutrient medium and accumulation of the nutrient medium are not so limiting. For example, to obtain 2 m ^{2 of} new tissue using a honeycomb matrix, approximately 250 tissue culture flasks would be required, typically 80 cm ² of free surface area per flask.

Preferably, dermal fibroblast cells are grown in a bioreactor until they reach the stage of fusion or approximate fusion with each other, in which the cells almost completely cover the microsphere. Cell fusion is usually achieved within 7 days. However, it is possible to use cell-coated microspheres therapeutically, and until that time, are in the process of approaching fusion, when the cell population is still in a state of high migration and rapid reproduction. The cells are then concentrated by removing excess culture fluid and washed in situ with an appropriate buffer or solution to obtain a microsphere / cell suspension. In contrast to conventional microcarrier cultivation practice, whereby a factor such as trypsin is then used to remove microcarrier cells, the dermal fibroblast cells are not removed from the microspheres according to the present invention. A suspension of microspheres with cells is then applied directly to the wound in the same way as a solution or ointment. Due to the uniformity of the suspension of microspheres, each microsphere has the same number of cells attached to it, which leads to a homogeneous distribution upon further application to damaged skin. Moreover, there is homogeneity of the cell growth phase in the area of skin damage, even in the case of a wide area of damage,
In contrast to the area of the cellular matrix described above, the invention reduces the number of manipulations with cells. This in turn reduces the likelihood of damage to the cells themselves and minimizes the likelihood of contamination leading to infection of the wound.

 Cells applied to the wound in accordance with the invention can be easily distributed over the entire surface of the wound. This is a very important advantage, since cells grown on a cellular matrix, and even skin for transplantation received from a patient, often have many non-contact areas in which they cannot be completely restored. The consistency of the suspension allows you to adjust the contour changes in the wound. This is a great advantage over planar implants according to the prior art. Firstly, the wound rarely has a uniform depth with a smooth flat base. Secondly, it can spread over a significant area. For example, it is clear to a specialist that when treating a burn formed along the entire length of the arm on its back, there are many natural bends along the entire length of the arm. The invention provides an implant that can fill even the deepest wound for its effective treatment and more natural three-dimensional tissue regeneration at the site of injury.

 An example of uneven damage to the skin layer throughout its thickness is shown in FIG. Damage to the skin 10 extends through the epidermis 11 and the dermis 12 to the muscle 13 located beneath it. One skilled in the art will appreciate that a flat skin graft will not provide an adequate skin implant for this type of damage. Figure 4 schematically shows the same damage to the skin 10 with the implant implant of healthy skin substitute 20 according to the invention.

 Healthy skin substitute 20 creates an implant of dermal fibroblasts 21 and keratinocytes 22. Serial layers of dermal fibroblasts can be applied to the wound over a layer of dermal fibroblasts, for example, to correct any further changes in the contour. It is also possible to apply to the wound a suspension obtained from a donor of dermal fibroblasts with microspheres, followed by applying to it a suspension obtained from a patient of dermal fibroblasts with microspheres.

 The use of cells under conditions of fusion or close to fusion on microspheres not only provides a high concentration of cells at the site of skin damage, but also increases the efficiency of cells. Animal cells tend to function better if they are in a microcosm, such as found in a fusion, or go close to a fusion of a cell population on the microsphere. It is also achieved by improving the production of growth factors and distribution, especially in cells that are in close contact with each other as a result of intercellular regulation, including autocrine and paracrine interactions, reducing diffusion restrictions, and so on. The application of the dermal layer allows you to quickly obtain structural protein collagen and other growth and binding factors. Earlier application of dermal fibroblasts can reduce or prevent skin contraction, which is the main cause of scarring and loss of excess fluid. Dermal fibroblasts can differentiate and combine axially in the wound to effectively connect the wound, especially if the skin lesion is a longitudinal incision. Earlier application of microspheres coated with fibroblasts leads to better healing of the wound and reduces the suffering of the patient.

 After applying the suspension to the wound, the wound area may be covered with a gas-permeable dressing. Dermal fibroblasts in suspension with microspheres with cells then migrate and grow in the direction from the microspheres to the surrounding tissue and create a continuous surface at the site of skin damage. After some time, when the cells grow, the microspheres begin to resorb in vivo.

 Preferably, the skin implant also includes regeneration of the epidermal layer. While the dermal fibroblast layer is created in vivo, the cells of the patient's epidermal layer can be cultured in a tissue culture flask. After obtaining a sufficient number of cells, the cells are removed from the bottom of the tissue culture flask using, for example, trypsin. Cells are then seeded in a bioreactor containing a suitable culture medium and microspheres. As described above, another optimal option is to use epidermal cells from a tissue explant obtained from a patient to directly inoculate microspheres, cells are fixed on microspheres and cultured in suspension in a bioreactor in the same way as previously described in the case of dermal fibroblasts. The specialist understands that it may be necessary to use a wide range of antibiotics in the initial phase of cell reproduction due to the possibility of infection of the wound area with flora from healthy skin and from other sources.

 Figure 5 shows photomicrography obtained from the placenta of newborn keratinocytes attached to PHB-PHV microspheres, 24 hours after plating of cells. Most keratinocytes attached to the microspheres became flat and lost their spherical shape. As mentioned above, microspheres can be added to the culture medium just before seeding cells, or they can be pre-impregnated in this medium for some time before seeding cells. Figure 5 shows a micrograph of keratinocytes obtained from the placenta of a newborn (neonatal foreskin) and attached to PHB-PHV microspheres, 24 hours after plating of cells. The microspheres were incubated in the culture medium for 24 hours before plating the cells. Most keratinocytes attached to the microspheres took a flat shape.

 After epidermal cells, including keratinocytes, have reached fusion or approximate fusion on the microspheres, part or all of the culture medium is removed and a suspension of cell-coated microspheres is obtained. Dressings, if any, are removed from the wound and a suspension is applied over a layer of dermal fibroblasts to regenerate the outer layer of the skin, thereby creating a protective barrier to intact skin.

 After applying a suspension of epidermal cells to the wound, the site of damage can be covered with a gas-permeable dressing. The epidermal cells then migrate and grow in the direction from the microspheres to the surrounding tissue to create a continuous surface for the substitute for skin damage. As the cells grow, in vivo resorption of the microspheres occurs. A certain percentage of epidermal cells can be treated either in vivo or in vivo with a specific factor, such as calcium and / or CAMP, to accelerate the production of stratum corneum, the topmost layer of dead highly keratinized cells. Stratum corneum helps regulate the amount of water lost by the body, as well as prevent the invasion of germs in the wound area. With a controlled acceleration of the differentiation of this layer, the healed wound site will function like intact healthy skin.

 According to another embodiment of the invention, cells of both the dermal and epidermal layers are cultured in a tissue culture flask. This embodiment of the invention makes it possible to develop enhanced paracrine and autocrine functioning. Using the same technology as previously described, the cells are then removed from the tissue culture flask and seeded in a bioreactor containing the culture medium and microspheres. Cells are fixed on microspheres under static or semi-static conditions for subsequent co-cultivation in suspension in a bioreactor. An individual microsphere can then have cells of both the dermal and epidermal layers attached to it. The resulting suspension of microspheres with cells is applied directly to the wound in the same way as an ointment or paste is applied to the wound to restore both the dermal and epidermal layers.

 As it happens in vivo, differentiating keratinocytes tend to migrate to the highest places of the wound, which leads to the natural formation of stratum corneum. Then the wound can be covered with a gas-permeable dressing until its effective healing as a result of the natural transformation of the dermal and epidermal components.

 According to another embodiment of the invention, dermal fibroblasts and cells obtained from the epidermal layer are cultured independently in separate tissue culture flasks. Dermal fibroblasts are removed from the corresponding tissue culture flask using, for example, trypsin, and they are seeded in a vessel containing the appropriate cell culture medium and microspheres. Then dermal fibroblasts are fixed on the microspheres in static or semi-static conditions.

 Similarly, epidermal cells, including keratinocytes, are removed from the corresponding tissue culture flask using, for example, trypsin, and they are seeded in another vessel containing a suitable cell culture medium and microspheres. Then the epidermal cells are fixed on the microspheres in static or semi-static conditions. Microspheres coated with dermal fibroblasts and microspheres coated with epidermal cells are then introduced into the same bioreactor for subsequent co-cultivation of them.

 The resulting suspension of cell-coated microspheres is then applied to the wound to simultaneously restore the dermal and epidermal layers. The wound may then be covered with a gas permeable dressing.

 According to another embodiment of the invention, dermal fibroblasts are fixed on microspheres and subjected to rapid propagation in a bioreactor. After some time, the cells are sown from the epidermal layer in the bioreactor for subsequent attachment to microspheres previously covered to some extent with dermal fibroblasts. As a result, keratinocytes may benefit from the paracrine effects of fibroblasts.

 The invention can also be used for in vivo implantation of melanocytes in order to impart natural pigmentation to the skin and to protect against UV.

 In the field of cosmetic medicine, for example for treating scars for wounds, acne, etc., the epidermal layer and a predominantly small percentage of the dermal layer below it can be surgically removed with a dermatome or other suitable surgical instrument. After that, a suspension of cell-coated microspheres in one or more layers is directly applied to the pre-treated area to correct contour changes.

 The microspheres used according to the invention can be made of various materials that are biocompatible and capable of resorption into the body floor by exposure to in vivo natural enzymes without the formation of toxic by-products.

 Suitable materials for microspheres are natural or synthetic bioresorbable materials, such as polyhydroxybutyrate (PHB), copolymer PHB - polyhydroxyvaleriate (PHB-PHV), polyester-linked PHB, polylactidoglycolides, lipids, phospholipids, polylactones, polyesters, polymeric polyolactides , gelatin or other resorbable materials that do not adversely affect the tissue during treatment (i.e., are not toxic to the cells initially or through the end products of resorption). These materials can be used in pure form or as a mixture of materials to improve physicochemical properties or to control the rate of their decomposition.

 Preferably, the microspheres have a density of from 1.01 to 1.04 g / ml to facilitate mixing and suspension in a culture medium. The microspheres can be relatively equal or with a changing surface and have a microporosity of 30 to 80% with a pore size of 30 to 80 microns.

The microspheres have a relatively large surface area, for example, about 5000 cm ² / g dry weight. For comparison, cell matrices according to previously known planar technology typically have a surface area of 200 to 700 cm ² / g. Moreover, to achieve adequate strength, a greater consumption of material for the manufacture of a cellular matrix is necessary. Accordingly, much more material will be needed to obtain implants with the same surface area, and therefore, more material must be resorbed in vivo when using a cellular matrix.

 It will be apparent to one skilled in the art that other structures, such as cylindrical, ovoid, wafer-like, can be used in the substitute for living skin according to the invention.

 Microspheres can consist of a core of one type of material with a shell in the form of an outer layer or a coating of another material to improve resorption and functional properties, including charge density, the ability to adhere other chemicals or components, and improve cell adherence and distribution. For example, phospholipid coating promotes the formation of a polar surface with functional phosphate groups. The acyl groups of phospholipids create a more hydrophobic surface of the microspheres. Various chemicals and biomolecules can then be attached to this part of the phospholipids. In addition, the microspheres may be coated with certain stratum corneum lipids, including phospholipids and sphingosines to improve antimicrobial activity. Sphingosines are especially good for inhibiting microbial reproduction at microgram levels (Antimicrobial activity of sphingosines, J. Invest Dermatol, 98: 269-273, 1992).

 In FIG. 7 shows a histogram of data obtained on a cytofluorometer for measuring cell viability. Cell viability is measured using fluorochrome specifically for living cells and fluorochrome specifically for dead cells.

 The measurement results are expressed as "Conditions of fluorescent elements" and applied to the histogram in the form of crosses denoting living cells, and in the form of dashes representing dead cells. The interpretation of the data is adjusted by the data set 1 and 2, respectively, for the phosphate buffer solution and for the control. Keratinocytes from the same cell colony were seeded on phospholipid coated PHB and PHB and, in each case, incubated for 24 hours. The viability of the attached cells on the respective samples was measured using the fluorochromes described above. Samples without cells attached to them were also measured on fluorochromes for additional correction taking into account the specificity of the resorbed material. Cell-free samples are represented by data set 3,5,7 and 9 as corrective factors for the data set 4,6,8 and 10, respectively.

 Data set 4 and 8 represents the viability of keratinocytes attached to PHB, while data set 6 and 10 shows phospholipid-coated PHBs. The resorbable materials corresponding to dataset 3,4,5 and 6 were autoclaved before seeding, while the resorbable materials corresponding to dataset 7,8,9 and 10 were sterilized without heat treatment with ethylene oxide. Data indicate high cell viability on these resorbable materials. Moreover, the method of pre-sterilization (steam or ethylene oxide) does not affect the efficiency of fixing or viability of the cell population. According to another embodiment of the invention, the microspheres are obtained from a mixture of a polysaccharide and a resorbable material, so that the resorbable material is unevenly distributed over the mass of the core. A polysaccharide (such as dextran or starch) can be destroyed or removed by chemical or enzymatic means before coating the microspheres with skin cells, leaving the structure open for hydrolysis of the resorbable material in vivo. This type of microsphere reduces the diffusion limitations of nutrients or intermediate metabolic products, which further accelerates cell growth. In addition, various factors, for example, growth factors and others introduced inside or onto the surface of the microspheres, are removed more quickly, which facilitates access to these factors at a certain time during the treatment period.

 Alternatively, microspheres can be used with an intact polysaccharide component for resorption during rapid cell propagation in vitro and in vivo.

 In a preferred embodiment, the microspheres resorb at a rate comparable to the spread of a cell population. Thus, as the microspheres are resorbed, the cells grow inside small voids formed by the partial removal of the resorbable microspheres, thereby minimizing the formation of a scar and changing the contour.

 One approach to selecting materials and / or factors or coatings is to choose one combination for the dermal layer and another for the epidermal layer. Preferably, the microspheres in the epidermal layer are resorbed much faster than the lower material for the dermal layer. This contributes to a more natural migration and the spread of keratinocytes to the dermal layer below. It is also possible to use different coatings for different types of cells, although one type of coating may be suitable. Microspheres can also have other properties that are given to them by introducing additives inside or onto the surface of the microspheres, for example, by immobilization, encapsulation, covalent attachment, or simple adsorption. These additives are used to accelerate cell growth, to improve the local environment in vivo, followed by implantation and / or to control the removal of a specific factor. Such additives can be introduced inside or on the surface of almost all microspheres of the implant, or only part of them.

 For example, the controlled entry of antimicrobial factors is especially important, since many of them are cytotoxic both to dermal fibroblasts and to keratinocytes, which creates a negative effect on wound healing. Improvements in the controlled entry of the antimicrobial factor are achieved by pretreatment of the microspheres with the antimicrobial factor, which leads to a less negative effect on wound healing in the case of aseptic content.

 Alternatively, the surface of the microspheres can be pretreated to facilitate cell attachment to them, for example, by a fixing factor such as arginoglycine aspartic acid tripeptide (RGD) or poly-L-lysine. Fig. 8 shows photomicrography showing dermal fibroblasts obtained from neonatal foreskin placenta and attached to PHB-RHV microspheres treated with RGD as a fixing factor 1 hour after cell plating. Fig. 9 shows photomicrography showing the same dermal fibroblasts attached to RGD-treated PHB-PHV microspheres as in Fig. 8, but 22 hours after cell plating. Most of the cells that are attached to the microsphere have turned from spherical to flat.

 Figure 10 shows photomicrography of dermal fibroblasts obtained from neonatal foreskin placenta and fixed to a glass surface (control experiment) after 26 hours of incubation. 11 shows photomicrography of dermal fibroblasts obtained from neonatal foreskin placenta and attached to the planar surface of PHB-PHV after 26 hours of incubation. 12 shows dermal fibroblasts obtained from neonatal foreskin placenta and secured to a flat surface from RGD-treated PHB-PHV and incubated for 26 hours.

 Living cells in FIG. 10-12 are marked with Neutral Red. The dye is actively selected by the cells to illustrate in FIGS. 11 and 12 that the cells on the bioresorbable materials used are highly viable.

 Other factors that can be introduced into the microspheres include growth factors such as epidermal growth factor (EGF), transforming growth factor-alpha (TGA), transforming growth factor-beta (TGF-beta), keratinocyte growth factor (KGF), basic fibroblast growth factor (bFGF) and insulin-like growth factor-I (TGF-1). The controlled entry of these factors can be achieved by using microspheres to better control wound healing, because if the mitogenic factor (growth factor) or angiogenic factor (causing recapillarization of the wound) is too fast, a scar may form at the site of the wound. Growth factors can also be immobilized on the surface of the microspheres with the formation of a constant covalent bond, so that the cells come into contact with the immobilized growth factor.

 Monoclonal antibodies can be attached to the microspheres to regulate the local in vivo level of TGF-beta, for example, to avoid excessive supply of this factor in a certain place during wound healing. The intake of the usual amount of TGF-beta can lead to a disproportionate excessive reproduction of collagen by fibroblasts and macrophage cells.

 The resorption of microspheres is facilitated by the introduction of enzymes into the microspheres, such as lipase, depolymerase and dehydrogenase. These enzymes promote resorption by other endogenous in vivo enzymes or free radicals generated by the body (for example, macrophage cells) that can attack structural bonds, resulting in depolymerization of the bioresorbable material.

 It is also possible that some substances attached to or introduced into the microspheres can be obtained directly from the patient. For example, growth factor derived from blood platelets (PDGF) (known as wound healing aid) can be obtained from a patient’s blood platelets and attached to or on the surface of microspheres. This requires a relatively small blood sample, for example, 100-200 ml.

 Other additives include: polyamines that reduce hypertrophic scars; substances that impart biostatic, microbicidal and anti-rejective properties, and proteinoids that increase activity and / or stability.

In addition, growth, migration, angiogenesis, and other factors can be introduced inside or onto the surface of additional microspheres, on which cell deposition is not intended. For this purpose, it is possible to use ultrafine microspheres, since it is not required to use them in large quantities, thereby reducing the amount of material to be resorbed. These ultra-small microspheres with a diameter of 10 to 50 microns can be distributed together with large cell-coated microspheres in the wound area or introduced additionally at the right time.
The surrounding tissues can be treated with chemotactic, angiogenic and mitogenic factors that cover the surface of such cell-free microspheres to improve the environment for cell migration, nutrient delivery, migration and growth. Another advantage of the invention is that in spite of the relatively low stability of these factors in solution, they can be prepared fresh immediately before treating the wound with microspheres coated with cells. These microspheres can be distributed and served simultaneously with or before the cells in order to facilitate cell migration, angiogenesis and / or rapid reproduction. Thus, a programmable environment can be created for accelerated tissue regeneration and migration to the wound area. As a result, the lower capillary network can be enlarged for the “normal” nutrition of the applied cells.

 Significant successes have been achieved over the past few years in creating a culture medium that does not contain animal serum. This is especially important for technologies intended for use in clinics for people. Serum, usually obtained from bovine sources, is not only completely uncertain, but can also serve as a source of contamination of the final product and lead to variable results. A specific serum-free medium is suitable for culturing dermal fibroblasts and keratinocytes. Many serum-free culture media are available in industry, for example, Clonetics, Corporation KGM, FGM and GIBCO KGM.

 In the process of co-cultivation of keratinocytes and dermal fibroblasts, keratinocytes require a lower calcium content in the medium for rapid growth and to prevent final differentiation. With a high calcium content (> 0.1 mM), keratinocytes tend to slow growth and undergo a final differentiation leading to apoptosis (programmed cell death).

 Dermal fibroblasts are usually grown in a medium with a calcium content of 1 to 3 mM, which ensures their normal growth and functioning, respectively, according to one embodiment, dermal fibroblasts are first cultured in a medium with a calcium concentration of more than 0.1 mM. After some time, the cells are washed with a solution of phosphate buffer (PBS) in situ to remove the growth medium. Then, keratinocytes and another medium with a low calcium concentration are added to a tissue culture flask containing cultured dermal fibroblasts. The result of further cultivation is a decrease in the growth of fibroblasts and good growth of keratinocytes. Such conditions are ideal since they promote the growth of the keratinocyte layer in the presence of fibroblasts without the growth of fibroblasts on top of keratinocytes. Although fibroblasts do not multiply rapidly, they can produce the factors required by keratinocytes for optimal growth and construction of the dermal surface. Factors produced by fibroblasts that promote optimal growth / organization of keratinocytes include fibronectin and TGF-beta.

 The invention creates the potential for the geographical centralization of agents for treating wounds or burns in cases of effective cell manipulation. Applying dermal fibroblasts in case of damage to all or part of the skin layer can be done more quickly when creating a local "bank" received from a donor, including deceased, dermal fibroblasts, which exhibit low allergenicity in transplants.

The tissue explant obtained from the patient can then be transported in an appropriate transport medium similar to that used for transporting organs for transplantation (such as ViaSpan (trademark) UW Solution, Dupont or other preservation medium) to a cell dilution center for keratinocyte culture, while the patient will already be treated with a suspension of dermal fibroblasts with microspheres. Typically, a 4 cm ² tissue explant can be quickly transported and expanded if necessary to sizes sufficient to process an equivalent tissue area of several square meters in size for one to three weeks.

 The growth of a sufficient number of cells for processing occurs much faster and more efficiently than with the usual approach described above.

After reaching the required cell size, populations coated with microsphere cells can be collected, concentrated and placed in a preservation medium for delivery to a remote processing center. In a relatively small volume, a large number of cells, on the order of 10 ⁷ cells / ml, can be transported on microspheres. This is an advantage over planar technology, requiring a large number of containers for an equivalent number of cells. In addition, the transportation of the cellular matrix is complicated by the fragile nature of the preparation of the fabric. The suspension of microspheres with cells according to the invention is less fragile and can nevertheless satisfactorily transfer transport under far from ideal conditions.

 Cell-coated microspheres can be transported in a medium similar to that used to transport organ transplants, such as the heart. Moreover, the medium can be adapted to include perfluorocarbons for better oxygen transfer. Suspension of microspheres with cells either does not require at all, or requires a little additional processing before applying to damaged skin. Known technologies such as tangential membranes or hollow fiber membranes retaining microspheres with cells during manipulation of the medium in which they are located can also be used to transport them.

 The previously described cellular matrix implants are typically cryopreserved before transport. However, it is clear to a person skilled in the art that the cryopreservation medium may contain dimethyl sulfoxide (DMSO), which requires complex techniques, including a sterile environment, and many operations, which requires a tissue culture laboratory to obtain and to remove before implantation. This type of service is not always available on site when or where processing is needed.

 The invention allows for quick and effective treatment of damage to all or part of a layer of damaged skin, for example, for burns and other wounds. In contrast to conventional methods, the invention does not require sutures or brackets or other methods of fastening when applied to a patient. It is not fraught with problems associated with the configuration and bending of the body. Moreover, the application of a suspension of microspheres with cells can be carried out both under ideal and far from ideal conditions.

 The substitute for living skin according to the invention can also be used as a “skin model” for hazardous and safe evaluation tests of substances for the pharmaceutical, cosmetic and household industries. This area is expanding due to the demands of cunning animal testing.

 The invention can also be used in cosmetics to correct skin defects.

Claims (26)

 1. A substitute for living skin, comprising a biocompatible carrier and a culture of skin cells, characterized in that it contains, as a biocompatible carrier, microspheres of in vivo resorbing material having an average diameter of 50 to 500 μm, preferably 80 to 250 μm, and a skin cell culture as a coating deposited on the surface of these microspheres.  2. The substitute according to claim 1, characterized in that as a culture of skin cells, it contains cells derived from the dermis or epidermis, or a combination thereof.  3. The substitute according to claims 1 and 2, characterized in that it contains skin cells obtained from a patient with an injury to the skin.  4. The substitute according to claims 1 and 2, characterized in that it contains skin cells obtained from a donor other than a patient with an injury to the skin.  5. The substitute according to claim 1, characterized in that it contains microspheres of at least one material selected from the group: polyhydroxybutyrate, a copolymer of polyhydroxybutyrate and polyhydroxyvalerate, polymers of lactidoglycolides, lipids, polylactones, polyesters, polylactides, polyglucolides, polyanhydrides, collagen and gelatin.  6. The substitute according to claim 5, characterized in that the microspheres consist of a core made of one type of material, and a coating of another type of material.  7. The substitute according to claim 6, characterized in that the microspheres are coated with phospholipids or sphingosine.  8. The substitute according to claims 1, 2 and 5 7, characterized in that the microspheres consist of polyhydroxybutyrate or polysaccharide.  9. The substitute according to claim 8, characterized in that it contains microspheres in which, before coating with their cells, the polysaccharide is chemically destroyed or enzymatically removed.  10. The substitute according to claims 1 and 2, characterized in that the microspheres contain at least one additive selected from the group: enzymes, binding agents, antimicrobial agents, growth factors, migration factors, angiogenesis factors, monoclonal antibodies, polyamines, materials, conferring biostatic, microbiocidal or anti-rejecting properties and proteinoids.  11. The substitute according to claim 10, characterized in that it contains lipase, depolymerase or dehydrogenase as enzymes.  12. The substitute according to claim 10, characterized in that it contains a platelet obtained from a patient or donor, or an epidermal growth factor, or a transforming growth factor alpha, or beta, or an insulin-like growth factor, or keratinocyte growth factor, or fibroblasts.  13. The substitute according to claim 10, characterized in that as the fixing agent it contains an arginine glycine aspartic acid tripeptide or poly-L-lysine.  14. The substitute according to claims 1, 2 and 10, characterized in that it contains additional microspheres having an average diameter of 10 500 μm, preferably 10 50 μm, containing additives inside or on its surface.  15. A method of producing substitutes for live skin by culturing skin cells and securing them on a biocompatible carrier, characterized in that microspheres of in vivo resorbable material having an average diameter of 50 to 500 μm, preferably 80 to 250 μm, are used and the cells are secured to these microspheres are carried out in the process of culturing cells in a culture medium until the cells cover the surface of the microspheres, followed by concentration of the resulting suspension by removing the culture fluid.  16. The method according to p. 15, characterized in that the use of cells obtained from the dermis, or epidermis, or combinations thereof.  17. The method according to PP.15 and 16, characterized in that when using a combination of dermal cells and epidermis, the process of culturing and fixing on the microspheres of the dermal cells is carried out in the first medium containing calcium with a concentration of more than 0.1 mmol, followed by washing with buffer a solution, culturing and fixing epidermis cells on these microspheres in a second culture medium containing calcium in a concentration of less than 0.1 mmol.  18. The method according to PP.15 and 16, characterized in that the cultivation and fixing on the microspheres of cells obtained from the dermis or epidermis, is carried out independently from one another.  19. The method according to PP. 15 and 16, characterized in that the use of a growth medium that does not contain serum.  20. The method according to PP.15 to 19, characterized in that the use of microspheres obtained from at least one material selected from the group: polyhydroxybutyrate, a copolymer of polyhydroxybutyrate and polyhydroxyvalerate, polymers of lactidoglycolides, lipids, polylactones, polyesters, polylactides, polyglycolides, polyanhydrides, collagen and gelatin.  21. The method according to PP.15 to 19, characterized in that the use of microspheres containing at least one additive selected from the group of: enzymes, binding agents, antimicrobial agents, growth factors, migration factors, angiogenesis factors, monoclonal antibodies, polyamines, materials giving biostatic, microbiocidal or anti-rejecting properties and proteinoids.  22. The method according to claim 20, characterized in that the additives are introduced into the composition of the microspheres or applied to the microspheres before coating them with skin cells.  23. A method of treating injuries of live skin by applying a substitute for living skin on the area of skin lesions, characterized in that as a substitute for skin, a substitute for live skin is used according to claims 11 to 14, which is applied as a suspension of microspheres coated with cells to the area of skin damage.  24. The method according to p. 23, characterized in that initially a suspension of microspheres coated with dermal cells is applied to the area of skin damage, then a suspension of microspheres coated with epidermal cells.  25. The method according to item 23, wherein a suspension of microspheres coated with a combination of cells of the dermis and epidermis is applied to the area of skin damage.  26. The method according to PP.23 25, characterized in that the correction of contour changes is carried out, followed by applying to the skin lesion area a suspension of microspheres coated with cells.

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